L-Glutamate oxidase is an enzyme which catalyzes the following reaction:L-glutamic acid+O2+H2O→α-ketoglutaric acid+H2O2 +NH3.
The known species of L-Glutamate oxidase include those obtained through isolation and purification from Streptomyces sp. X-119-6, Streptomyces violascens, and Streptomyces endus (Agric. Biol. Chem., 47, 1323–1328 (1983), Chem. Pharm. Bull., 31, 1307–1314 (1983), Chem. Pharm. Bull., 31, 3609–3616 (1983), Eur. J. Biochem., 182, 327–332 (1989)).
These L-glutamate oxidases have the following characteristics in common: (1) being produced from microorganisms belonging to genus Streptomyces; (2) remarkably high substrate specificity to L-glutamic acid; (3) comparatively stable under variations in temperature and pH; and (4) being a flavin enzyme requiring FAD as a coenzyme. However, these glutamate oxidases significantly differ in molecular weight depending on the microorganisms from which they have been obtained. For example, an L-glutamate oxidase derived from Streptomyces sp. X-119-6 has a molecular weight of about 140,000 (heteromer: α2β2γ2, α=about 44,000; β=about 16,000; and γ=about 9,000); that derived from Streptomyces violascens has a molecular weight of about 62,000 (monomer); and that derived from Streptomyces endus has a molecular weight of about 90,000 (dimer).
L-Glutamic acid, which is a predominant ingredient that imparts a flavor (umami) to food, is added to foods, inter alia processed foods, and such use amounts to about 1,000,000 tons per year. Since the L-glutamic acid content of foods can be readily determined by means of a kit or a sensor employing L-glutamate oxidase, this enzyme is now indispensable in the field of compositional analysis of foods.
Meanwhile, in recent years, in the field of cerebral nerve science, attempts to analyze L-glutamic acid—an intracerebral nuerotransmitter—have been energetically pursued by use of a microdialysis and a microsensor in combination. Most enzymes employed in the sensor are L-glutamate oxidases. Thus, the enzymes have become of more and more importance.
However, production processes of L-glutamate oxidase are not necessarily simple. For example, the L-glutamate oxidase derived from Streptomyces sp. X-119-6 is difficult to produce through liquid culturing, and therefore, solid culturing is employed to produce the enzyme.
Therefore, if a gene encoding L-glutamate oxidase can be obtained by cloning, L-glutamate oxidase can be readily prepared through a recombinant DNA technique, and new characteristics which native L-glutamate oxidase does not possess can be imparted to the prepared L-glutamate oxidase. However, hitherto, neither analysis of a gene encoding L-glutamate oxidase nor the amino acid sequence of the enzyme has been reported.